Production of dihydrostreptomycin



Apnl 5, 1960 Kom NAKAzAwA ETAI- 2,931,756

PRODUCTION OF DIHYDROSTREPTOMYCIN Filed March 21. 1957 3 Sheets-Sheet, 1

K. NAKAY-Awn, M smhm, BY Krmxex HxAMAmoro www, EL

@W ATTN, S

PRODUCTION F DHYDROSTREPIOMYCN Koiti Nakazawa, Higashitomatsu, Amagasaki, Motoo Shibata and Kazuo Tanabe, Toyonaka, Osaka, and Hiroichi Yamamoto, Mikage, Mikage-c'ho, Higasltinada-ku, Kobe, Japan, assiguors to Takeda Pharmaceutical Industries, Ltd., Doshomachi, Higashi-ku, Osaka, Japan Claims priority, application Japan March 24, 1956 7 Claims. (Cl. 195-80) The present invention relates to the production of dihydrostreptomycin. l

Dihydrostreptomycin is a lvery useful substance for medical treatment because it shows a strong and wide antibacterial spectrum like streptomycin and is, in addition, less toxic and more stable than the latter. Moreover, dihydrostreptomycin, which resembles streptomycin in antibacterial properties, has the advantage over the latter that it exhibits much lower neurotoxicity. Dihydrostreptomycin has so far been obtained by hydrogenation of the carbonyl radical of streptomycin and placed on the market as its sulfate or hydrochloride. These salts are colorless or white crystalline, or powdery substances. They are odorless or nearly odorless, taste slightly bitter, and their aqueous solutions are levorotatory, and an aqueous solution of the crystal of the sulfate shows [a]D25=-88 (c=l). It is well known that dihydrostreptomycin shows a wide antibacterial spectrum against gram-positive, gram-negative and acid-fast bacteria.

As mentioned before, dihydrostreptomycin has been nited Sttes Patent 2,93L756 Patented Apr. 5, 1960 prepared exclusively by reducing streptomycin isolated from the culture broth of Slreplomyces grseus. etc. (U.S. Pat. No. 2,498,574), but this method requires highly pure streptomycin, otherwise a considerable loss by the reduction is inevitable.

The direct preparation of dihydrostreptomycin by the cultivation of dihydrostlreptomycin-producing microorganisms has not been published in any report prior to the present invention. Such a method will simplify the preparation of dihydrostreptomycin, enhancing the yield and lowering the cost, and the embodiment thereof is a desideratum in this art.

A primary object of the present invention is the development and embodiment of such a method for the direct preparation of dihydrostreptomycin by the cultivation of dihydrostreptomycin-producing micro-organisms. The present invention has realized the said object with the aid of certain micro-organisms (strains of Streptomyces) which, when cultivated under appropriate conditions, produce dihydrostreptomycin in the culture broth in collectible amount. Briefly stated, the method of preparing dihydrostreptomycin, according to the present invention,

is characterized by inoculating a dihydrostreptomycin- STRAIN 23572 Cultural characteristics Medium Remarks Growth Aerial myeelium and spores Soluble pigment Czapek agar Colorless White N one Glucoseasparagineagar. do White to smoke-gray (Rdg XLVI, --.do. Abundantly interspersed with small moist A 21" -d or Vinaceous-but (Rdg black patches which gradually spread XL, 17m-d). over the whole surface. Reverse Creambutt` (Rdg XXX, 19-d) or Cartridgebult (Rdg XXX, 19-f), later becoming Y Chamois (Rdg XX-X, 19"-b). Starch asm' dn White to pale smoke gray (Rdg do.. Reverse Cream-buff (Rdg XXX, 19"-b).

XLVI, 21""-f). Y Hydrolysis slight. Calcummalate agar..-, Colorless, later becoming Scanty white-. ..-do.....

t11))ut-yollow (Rdg IV, 19- Glycerin nitrate agar--. Colorle s -.-.-dn Dextrose nitrate agar. ...-.dn do Bouillon agar dn Nnm Gelatin .--dn o Moderate liquefaction. Potato plug` do Wzhite' to smoke gray (Rdg XLVI, Moist black patches observed. Carrot plug.. dn .-.do n Yeast extract agar do Wh7ilte' light drab (Rdg XLVI, Partially moistened.

l Whole egg dn White Milk dn do Peptonization slowly. Glycerin asparaginate ....do White to smoke gray (Rdg XLVI,

agar. 21"d). Peptone nitrate broth.. Nitrate reduction.

Aerial mycelium ol this strain shows spiral, 0.8-1.2, conidia oval. 11.5nxl.52.

Vthe -strains useful in the present invention.

, Carbon utilization of the strain No. 23572, measured by the Pridhams method, is as follows:

Dulstol...

dl-lnositol. Salicin.. Na-acetate.. N11-citrate Y Na nerinato good growth.

i=growth doubtful.

++=fair growth.

From the characteristics mentioned above, the species is a new species, and has been named Strepzomyces humidus nov. sp. Nakazawa et Shibata.

Taxonomical comparison of this species with Streptom'yces hygroscopicus, which resembles the former in property, is shown inthe following table:

kcoMPARrsoN BETWEEN sTREPToMYcEs .HU-

MIDUS AND STREPTOMYCES HYGROSCOPICUS Streptomyces hygroscopicus produces hygroscopin ac cording to J. Agr. Chem. Soc. (Japan) 28, 296 (1954) and Antibiotics and Chemotherapy 3, 1268 (1953), Carbomycin according to Antibiotics and Chemotherapy 3, 899 (1953), Angstmycin according to J. Antibiotics, Japan 7, 113 (1954), andHygromycin according to Antibiotics and Chemotherapy 3, 1268 (1953), whereas Streptomyces humidus produces dihydrostreptomycin.

The above-mentioned Streptomyces humidus strain is hygroscopic, but some strains of the same species have .no .such property. Also, some `form no aerial mycelium and others produce pigment.

The above strain represents only one example of the Strains belonging to other species also can be used for the same purpose so far as they produce dihydrostreptomycin as a metabolite, even if they do not much resemble Streptomyces humz'dus n'ov. sp. in property. l

As observed in micro-organisms, especially in Streptomyces, their behavior on the culture medium .easily changes spontaneously or may be changed artificially,

and therefore identification of a species is so diicult .that

a description of the properties of a species may not suflice for the identification of the species. Hence, the Ypresent invention includes, besides the above species, its `variants isolated from soil, mutants induced from them -by mutating agents 'such as X-ray, ultraviolet-rayY andche'rnicals, and any strains isolated from soil basing on the findings of the present invention soYf'ar as they fulfil the necessary requirements.

Taxonomical characteristics of some mutants induced from Strepmmyces humidus by the conventional mutating agents such as ultraviolet radiation and mono-spore separation are shown in the following table:

MUTANTS OF NO. 23572 23572 G 23572 Y 23572 W Glucose Asparagin Agar.. Tea- Starch Agar- Whitem.. Dextrose Nitrate Agar do Potato plug Glycerin asparaginate agar.

Pale orange-yellow. White.

.do Cream Color Do. Capucine-buil later Do.

orange-yellow. Pale orange-yellow. Do.

green. White-- Most strains satisfying the requirements of the present invention do not resemble Strepzomyces griseus taxonomlcally, and are resistant to both dihydrostreptomycin and streptomycin. Differing from the antibiotics obtained from streptomycin-producing strains, the crude antibiotics obtained from dthydrostrcptomycin-producing strains are generally negative to maltol reaction. These properties can be advantageously utilized for separation of the desired strains from other strains. For example, with the properties as a criterion, the desired strains can be selectively separated by the dilution method or by cultivation on a medium containing dihydro-streptomyctn.

lAs stated, dihydrostreptomycin is produced and accumulated, according to this invention, by the cultivation of: the said strain on a liquid medium under aerobic conditions for a suicient time. It is remarkable, and wholly unobvious and unexpected, that dihydrostreptomycin is produced as a metabolite by the cultivation of a micro-organism under aerobic conditions.

Various substances which are used as nutrient source for cultivation of general micro-organisms can be ernployed in the present method. As carbon source, starch, lactose, sucrose, dextrin, glycerin and maltose can for example be employed. And as nitrogen source, organic or inorganic nitrogen-containing substances such as soybean meal, meat extract, peptone, peanut powder, casein, amino acids, yeast, bran, corn-steep liquor, cotton-seed powder, nitrates, urea and ammonium compounds can be used. Also a small amount of inorganic salts and 'growth-promoting substances maybe added to the medium. As other nutrient sources mycelia of a strain belonging to lenicilliurn or its culture `broth may for instance be used. Any culture media suitable for the cultivation of Slreptomyces griseus were found to fulfil the same purpose in the instant process. A precursor may also be added if necessary.

The culture medium may be solid or liquid, but an aerobicsubmerged culture is preferable for industrial purpose.

When'Streptomyces humdus is used as the dihydrostreptomycin-producing micro-organism and the cultivation is conducted under aerobic submerged conditions, the cultivation may preferably be conducted at a tempera.- ture of about 24 to 30 C. over a period of 3 to'8 days, but the temperature and the period must be adjusted according to the other culture conditions. And, of course, when another micro-organism is used, the most preferabe conditions for its nature should be employed.

In the case of liquid culture, the accumulation of dihydrostreptornycin can attain several ten to several thousand micrograms per cubic centimeter, but vthe dihydrostreptomycin can be effectively separated even from an about lO'y/cc. solution.

The dihydrostreptomycin may be isolated and purified ina variety of ways, advantageously after themanner set forth in application, VSerial No. 647,682, filed on even date herewith, e.g. by filtering the fermentation broth to remove solid culture materials or substances precipitated after the cultivation- The `active compound in the 'iiltrate is adsorbed 'in acatiou-echange and' then eluted with an acid solvent. Thus a concentrated solutron of the active compound containing a small quantity of impurities is obtained. The solution is further concentrated if necessary and subjected to adsorption chromatography to eliminate almostall the impurities.

The dihydrostreptomycin thus obtained is characterized by the following:

(A) Physical and chemical propertiesV (1) Infra-red absorption spectrum was observed on the following derivatives induced from the product of the present invention (referred to as Antibiotic 23572 hereinafter) and commercial dihydrostreptomycin:

I. Sulfate, II. Streptidine picrate, III. a-Methylpentaacetyldihydrostreptobiosaminide.

As shown in the accompanying drawings, i.e. Figs. 1, 2 and 3 (correspond to I, Il and IH respectively), al1 the spectra were in complete accord with those of authentic samples, respectively. In the drawings, the curve drawn inv solid-line shows the spectrum of a derivative of Antibiotic 23572 and that drawn in dotted-line shows that of a derivative of dihydrostreptomycin, respectively. All the spectra were measured in Nujol mull with sodium chloride prism. i l

(2) Ultraviolet spectra of sulfate of Antibiotic 23572 and an authentic sample of dihydrostreptomycin sulfate were in good agreement,` and no specific adsorption was observed. Y

(3) Analytical value of the sulfate of Antibiotic 23572Y is in accord with the theoretical value of dihydrostreptomycin sulfate.

The theoretical value for dihydrostreptomycin sulfate [C21H41O12N7)2.3H2SO4]: C, 34.42; `I-I, 6.05; N, 13.38; S, 6.56. Found: C, 34.45, 34.11; H, 6.42, 6.54; N, 12.98; S, 6.43.

(4) The sulfate of Antibiotic 23572 shows [0:11325 87.4 (c=1, H2O), While dihydrostreptomycin sulfate exhibits [edDl1 -86.0.

The specific rotation of dihydrostreptomycin was reported as [u1D25 88 (I. A. Solomons et al.: Science 109, 515 (1949)) or as [:11325 88.5 IF. I. Wolf et al.: I. Arn. Chem. Soc., 68, 2163 (1946) 1. v

(5') The sulfate of Antibiotic 23572 melts at Z50-255 C. (with decomposition under blackeuing) and 'dihydrostreptomycin sulfate at Z50-255 C. (with decomposition under blackening) 1 (6) Both Antibiotic 23572 and dihydrostreptomycin v are negative to maltol reaction, but streptomycin is positive. Y

(7) Antibiotic 23572dihydrostreptornycin and strepvtomycin are all positive to Sakaguchi reaction.

(8) Solutions of sulfate of Antibiotie23572, dihydrostreptomycin sulfate and streptomycin sulfate in 0.1 .N-NaOH at a concentration of 5 mg./cc.,were left standing for 24 and 48 hours at room temperature, and the antibacterial potency against B. subtilis of each antibiotic -pwas assayed by the dilution method to give the following results:

immediately nfte . r dissolution, ullcc.

after 24- hrs., u.,'ec.

Sulfate of Antibiotic 23572 Dihydrostreptonrvcin sulfate streptomycin sulfate 1o. non 15, ooo 1o, 000

"6 antibacterial potency against B. subtilis 'of each'antibiotic was assayed by the dilution method to give'the following results:

30 mgJcc. solution of semicarbazidc shows potency of 75 u./cc.

(10) 1 ing/cc. of cysteine was added to each of the aqueous solutions of the sulfate of Antibiotic 23572, dihydrostreptomycin sulfate and streptomycin sulfate, and after standing at room temperature for 4 hours the antibacterial potency against B. subtilis of each antibiotic was assayed by the dilution method of give the following results:

4 hours no cysteine after is added, addition of u./cc cysteine,

u./cc.

Sulfate of Antibiotic 23572 (5 mg.,/cc.)-- 15. 000 15.000 Dihydrostreptomycin sulfate (5 mg./ec.) 15.000 15.000 streptomycin sulfate (5 mg./cc.) 15,000 7, 500

1 mgJec. solutionV of cysteine shows potency of 100 u./cc.

The sulfate of Antibiotic 23572 doesnot lose its activity by the addition of semi-carbazide or cysteine, as in the case of dihydrostreptomycin sulfate.A

(ll) Qualitative reaction for carbonyl radical is negative as shown below:

Fehling Phenol-conc. Silver mirror Reagent H2304 reaction Sulfate oi Antibiotic Negative No color Brown, clear.

23572. ization Dihvdrostreptomycln .do do Do. sulfate. streptomycin sulfate.-. Brown pre- Deep brown Blacky brown cipitate. color. precipitate.

(.12) Svtreptidine picrate was obtained from Antibiotic 23572 by the method of F. H. Stodola et al. (I. Am. Chem. Soc., 73, 2290 [195]) as follows: 2 g. of the hydrochloride of Antibiotic 23572 was dissolved in` cc. of anhydrous methanol containing 1% of hydrogen chloride. After standing for 48 hours at room temperature, 200 cc. of ether was added to the solution with stirring and the resulting white crystals (streptidine hydrochloride) Were separated by centrifugation. The crystals were dissolved in 20 cc. of water and a saturated picric acid solution was added. After standing for several hours the precipitated crystals were separated and recrystallized from Water to give 400 mg. of streptidine picrate as yellowish needles melting at 27 l-273 C. Analysis.- Calcd. for C8H18O4N6.2C5H3N3O72 C, H, N, 23.33. Found: C, 33.46; H, 3.44; N, 23.49. Infrared absorption spectrum of the picrate was in accord with that of the picrate prepared from dihyd'rostreptomycin as described in (1).

The supernatant of the above streptidine hydrochloride was neutralized with methanolic solution of sodium hydroxide, the resulting sodium chloride was separated by centrifugation, and the solvent was distilled off under reduced pressure, leaving white powder. The residue was dissolved in 20 cc. of pyridine and 7 cc. of acetic anhydride was added. After standing overnight, water was added to the reaction mixture and the solvent was dis- :tilled oi. The residue was recrystallized from methanol to obtain a-rnethylpentaacetyldihydrostreptobiosaminide,

colorless needless, M.P. 194.5 C, The yield was 700 mg. AfllySiSCalCdfor C13H19NO8(CH3CO)5(OCH3`): C, 51.15; H, 6.62; N, 2.49. Found: C, 51.09; H, 6.73; N, 2.40 [a]D1"=-l20.0 (C=1%, chloroform). Infrared absorption spectrum of the resultant was in good agreement with that of a-methylpentaacetyldihydrostreptobiosaminide' obtained from commercial dihydrostreptomycin as described in (1).

-Methyl pentaacetyldihydrostreptobiosaminide was obtained from Antibiotic 23572 by the method of N. G. Brink (J. Am. Chem. Soc., 68, 2557 [1946]) as follows: 10 g. of the hydrochloride of Antibiotic 23572 was dissolved in 500 cc. of methanol containing v1% of hydrogen chloride. After standing overnightVV at room temperature, the solvent was distilled off Yunder reduced pressure. The residue was dissolved in 750 cc. of methanol and 450 cc. of ether was added. The solution was passed through a column packed with 210 g. of acidwashed alumina impregnated with a 2:1 methanol-ether mixture. The column was then washed with 1000 cc. of a 3:2 methanol-ether mixture. The effluent was evaporated to dryness under reduced pressure. The residue was acetylated by being allowed to stand overnight at room temperature with 10 cc. of acetic anhydride and 10 cc. of pyridine. Water was then added and the solution was evaporated to dryness in vacuo. The residue was dissolved in chloroform and the chloroform solution was washed with water, dilute sulfuric acid and water; successively. After the chloroform was distilled the brownish solid residue was boiled for about 2 minutes with 100 cc. of ether, and the ether solution was separated by decantation. The same process was repeated again. The ether insoluble fraction was crystallized from methanol, yielding tat-methyl pentaacetyldihydrostreptobiosaminide, M.P. 194.5 C. The ether solution was concentrated to about 50 cc. and petroleum ether was added, giving white crystals. Recrystallization from methanol gave about 100 mg. of -methyl pentaacetyldihydrostreptobiosaminide, M.P. 155-156 C. No depression of the melting point was observed, when mixed with the -isomer prepared from dihydrostreptomycin as described above.

Analysis.-Calcd. for C13H19NOB(CH3CO)5(OCH3): C, 5l.l5; H, 6.62; N, 2.49. Found: C, 50.71; H, 6.62; N, 2.65. [a]D`-6=-36 (c=1%), chloroform). Infrared spectrum of the resultant was in good agreement with that of -rnethyl pentaacetyldihydrostreptobiosaminikde obtained from dihydrostreptomycin.

A s olution of 260 mg. of a-methyl pentaacetyldihydrostreptobiosaminide obtained from Antibiotic 23572 in 20 cc. of 10% hydrochloric acid was refluxed for three hours. After cooling, the brownish solution Was decolorized with charcoal and evaporated to dryness in vacuo. The residue was acetylated with 1 cc. of acetic anhydride and 3 cc. pyridine at room temperature. After addition of water, the solution was evaporated to dryness. The residue was dissolved in 50 cc. of a 7:3 benzene-petroleum ether mixture. The solution was passed through a column packed with 4 g. of acid-washed alumina impregnated with petroleum ether, whereupon the acetylation product was adsorbed into theV acid-washed alumina. The column was treated with 100 cc. of a 7:3 benzenechloroform mixture to elute the objective substance. The eflluent was concentrated to about 10 cc. and 30 c c. of ether was Vadded to give crystals. Recrystallization from chloroform-ether gave 25 mg. of needles, pentaacetyl-N-methyl-L-glucosamine, M.P. S-159 C.

Analyss.-Calcd. for ClqHggNOw: C, 50.62; H, 6.25; N, 3.47. Found: C, 50.71; H, 6.14; N, 3.24. [alD15 =102 (c=0.7%, chloroform).V` infrared absorption spectrum of the resultant was in complete agreement with that of pentaacetyl-N-rnethyl-L-glucosamine obtained from dihydrostreptomycin as described. No depression of the melting point was observed when. mixed with pentaacetylfN-methyl-L-glucosamine obtained from dihydrostreptomycin.

(13 Antibiotic 23572 was hydrolyzed with alkali undersimilar conditions used for hydrolysis of hydroxy streptomycin, described by F. H. Stodola et al. in I. Am. Chem. Soc., 73, 2290 (1951). That is, a solution of 2 g. of the hydrochloride of Antibiotic 23572 in 40 cc. of l-N-NaOH was heated at C. for 3 hours. The reaction mixture was acidiiied with hydrochloric acid and worked up, but no substance positive to maltol reaction nor a substance extractable under acidic conditions could be obtained, i.e. in this experiment Antibiotic 23572 gave a different result from those of streptomycin and -hydroxystreptomycin The same experiment with dihydrostreptomycin also gave the same result.-

(14) To 50,000 y/cc. aqueous solution of Antibiotic 23572 was added a warm solution of'methyl-orange until no more precipitate was formed. The precipitate was filtered and recrystallized from methanol-water (1 :3) to obtain the helianthate as scales, M.P. Z22-225 C. (Potency: 340 streptomycin unit per mg.).

Analysis.-Calcd. for dihydrostreptomycin helianthate (C03H86021N16S3): C, H, N, S, 6.40. Found: C, 50.19, 50.84; H, 5.93, 5.95; N, 14.93; S, 6.80.

(15) Paper partition chromatography was tried on streptomycin and dihydrostreptomycin by F. H. Stodola et al. (J. Am. Chem. Soc., 73, 2290 [1951] using water saturated butanol containing 2% of p-toluene sulfonic Vacid and 2% of piperidine as the solvent, and both compounds were distinguished by bioautogram.

Following the same method, paper chromatography was conducted on the above two compounds and Antibiotic 23572, and the bioautograms were examined using B. subtilis as test micro-organism. As the result, Antibiotic 23572 gave an inhibition zone at the site corresponding to that of dihydrostreptomycin.

(16) The crystallographical constants of the sulfate of Antibiotic 23572 were observed in comparison with those of dihydrostreptomycin sulfate reported by F. J. Wolf et al. (Science, 109, 515 [1949]). The results are (B) Chemotherapeutic Veect i The growth inhibitory action of Antibiotic 23572 on streptomycin-sensitive H37Rv strain of human tubercle bacilli was nearly on the same level as dihydrostreptomycin, effecting inhibition of growth in a concentration of 1 to 27 per milliliter in vitro. But it showed no inhibition of growth of the streptomycin-resistant strain of human type tubercle bacilli. The anti-bacterial activity of Antibiotic 23572 on the tubercle bacilli in vitro was considerably high, so its chemotherapeutic effect on experimental tuberculosis of mice and guinea pigs was further investigated as follows:

(1) Chemotherapeutic eect on experimental tuberculosis of mice (l).-Mice were infected intraperitoneally with 0.1 mg. (wet weight) of the H37Rv strain of human tubercle bacilli suspended in physiological saline (viable unit 8X106). On the following day of the infection, treatment of these animals was started with 3.0 mg. of the sulfate of Antibiotic 23572 per day and 1.5 mg. of dihydrostreptomycin sulfate per day respectively. These drugs were injected subcutaneously once a day 18 times in total. Three weeks after the infection all the animals were killed, and a definite quantity each of finely ground pulmonary, hepatic and splenic tissues from each animal was inoculated on solid egg media for the cultivation Qi tubercle. bacilli- Countinethe @logies 0f 9 .Y "10 tubercle'bacilli that developed on the media 4 weeks later, SULFATE OF ANTIBIOTIC-23572 (3.0 MG.) GROUP the antituberculous activities of the Idrugs were estimated. 7 n" The results are as shown in the following table: AiiimaiNi, 4 5 6 7 s 9 io UNTREATED CONTROL GROUP 5 Lung u o l 43 9 o 16 6 Liver 0 0 l 2 0 1 0 AnimalNo 7 s 9,10 11 '12in' 14 15 Spleen-n l0 2 12 1f 11 7 0 gutes g gg g g '1g 1g 1g 3g 5g AS hewn iii the above-table, the therapeutic efectpf Spleei'i s e0 2 34 228 70 5 600 241 10 Antibiotic 23572 on experimental tuberculosis of mice was as remarkable as dihydrostreptomycin. DIHYDROSTREPTOMYCIN SULFATE (1 5 MG.) (3) Chemotherapeulc effect on experimental tuber- GROUP culosis of guinea pigs-Healthy guinea pigs which showed negative tuberculin reaction (OT, 1:10, 0.1 cc., A l 15 intracutaneously, 48 hours) were infected subcutaneousy mma No 3 4 5 6 7 8 9 10 ly with 0.01 mg. (wet weight) of the H37Rv strain of Luni "o 0 0 o 0 o human tubercle bacilli suspended in 0.5 cc. of physiologi- Lveio o 1 o o o v 0 o cal saline (viable unit 35 X105). About one month after Spleen 0 0 0 1 2 the infection all of these animals became positive to tu- 20 berculin. At this time, 5 animals selected at random SULFATE 0F ANTIBIOTIC 23572 (30 MGJ GROUP were autopsied, when the establishment of gross tubercu- Y lous lesions in the organs of each animal was confirmed. Animal No 2 3 4 5 e 7 s 9 1o Then, the remaining animals were divided into 4 groups and treated as indicated below. When the drugs had iuniz 0 0 o 0 0 0 0 0 0 25 been administered subcutaneously 30 and 60 times '(ca. S'ei'jj'j': (5i g il g 2 21) 1g g 10 weeks and 15 weeks after infection, respectively), several animals of each group were autopsied, and all As seen from the above table, subcutaneous administrathe remaining aiiimais were kiiiefi 6 feeks after the end tion of the sulfate of Antibiotic 23572 amounting to 54 of the treatment (2i weeiis afi. iiifeciioii) The aiiiouiii mg. showed remarkable therapeutic effect on the experi- 30 of gi-Osi iiibeiciiioiis iesioiis iii .Organs oi these aiiimais mental tuberculosis of mice, and that it is as eiective as was? esiliiiliaiedl] aii aifso ii deigiiiitef qiiaiiiiti' each oiriiie the subcutaneous injection of dihydrostreptomycin sul- .iegioiia yiiip iio e o .primary iii ectioii site ii'iiig? ivei fate amounting to 27 mg. and spleen o f every v.animal was finely ground and inocu- (2) Chemotherapeulic eyject on expemenlal tubriatefi .on soiid egg. media. for the'ciiiiivaiioii of tubeicie, culoss of mice (11).-Mice were infected intravenously 35 iiaciiiikaiid coioiiies which developed during 6 Weeks with 0.001 mg. (wet weight) of the H37Rv strain of incubaiioii were counted'. on the ground of these iin-d." human tubercle bacilli suspended in 0.25 cc. of physioings the chemoihiapeuiic. eifeci? of the drug on experi' logical saline (viable unit 24Xm4). on the Second day mental tuberculosis of guinea pigs were estimated. of the infection, treatment of these animals was started. 4o Medicatiom YThe dru s were administered subcutaneousl 18 times over a peiod of 3 Weeks as follows: y Group 1 siiifate of Antibiotic 23572, 1o ing/day,

All the animals were killed 25 days after infection and siiiiciiiaiieoiisiy i the chemotherapeutic eiect of the drugs was estimated as Groubp 2-Sulfalte 0f AntlblOlC 23572, 20 mg./day, described before. sii ciiiiiiieiiiis y 45 Y- Group 3-Dihydrostreptomycin sulfate, 10 mg./day, Medication: subcutaneously Sulfate of Antibiotic 23572, 15mg/day, 18 times, Group 4 .Unrreated control subcutaneously Sulfate 0f AmlblOlC 23572, 3-0 mg-/dayi 18 flmesi The numberV of colonies obtained from the'cultivation of Sl-lbCUfaUeOUSlY 50 tubercle bacilli is shown in the following tables: Dihydrostreptomycin sulfate, 1.5 mgJday, 18 times, l -Y f subcutamusiy NUMBER 0F CoLoNiEs 0F TUBERCLE BACILLI UNTREATED CONTROL GROUP AFTER 6 WEEKS CULTIVATION AnimalNDu. 6 7 8 9 10 n |12 13 14 55 [10 mg.ofnely groutg-ggttigilg'ggtneozcehlgilliver and spleen were L u 8 22 i 6 n .88 a .0 2 65 38 CULTIVATED'IETIFE g'tFME 0F AU'ioPsY-JUST BEFORE Liliiii- 192 ii g5 25 lib 31114 i4 132 3 4 A 0F TREATMENT' Spleen- 17 57 549 110 372 103 I s3 6oo 225V AnlmalNo 1 2 4 5 DIHYDROSTREPTOMYCIN SULFATE (1.5 MG.) 0 2 o 2 i GROUP 2 s i3 1 4 5, 142 6 mimmo 2 3 4 5 6 7 s s 1o 60 60 )600 690 Lung 7 e1 5 5s io o 44 1o o 65 Liver 3 i 0 o 2 o 2 o 5 CULTIVATEDAT THE TIME 0F AUTOPSYAFTER aoTIMEs spleen 31 63 95 0 9 u 43 Gs v MEDICATION A y (Group 4) SULFATE OF ANTIBIOTIC 23572 (1.5 MG.) GROUP Y g A' mimmo 2 3 4 5 6 7 8 9 w 7o AnimalNo 11. 12 13 14Y 15Vv 1.6 17

Lung 19 5 io 3s i 102 1 5 g (7) g gli 1i 8g ii Liver 8 0 l l l 2 4 0 0 -Spleen n- 2 yi9 91 55 n 10 spleen 155 29 7 14 75 29 48 16 76 Lymphiiqiie oY 141 51s 315 209 352 iss (GTOLP 3) (Group 3) AnimalNo 6 7 8 9 10 11V 1 2 Animal No.'.;-. 23 24 25` 26 27 o3 o o o o 5 Lung o o o o o 21 o o o o Liver... o 4 0 o o 0' 0 0 l 0 Spfean..- 47 30 0 0 56 11s 2 102 11o o Lymphuode 32 140 21 283 111 (Group 1) 1 5 6 7 8 l Amm 19 2J 21 3i 31 o a o o o Q 0 0 0 0. 0 3 0 o o o n 0 0 0 0 0 0 o o o o o 212 o 21 2s o o -o 1 9s v1110 7 6o 116 13 Q 0 19 13.4 49 A (Group 2) 20 AnimalNo 21 22 22 21 2 23 27 s 6 7 l s 9 1o 11 H 1 Eme g g g g g 3 11 i iver.- g g 8 8 Y g g 2 g sp1een. 3 o o o o o 87 0 0 o o 3. 0 0 0 Lymphnodc 1 30 2 10 18 2 35 ,3. 3`3 0 0.35 0.3525

` v Y l (4) Toxicity-The LD50 values in mice (CF1) of the CULTIVATED AT THE TIME OF AUTOPSY AFTER 60 TIMES MEDECATION (Group 4) 9 1 134 1o 1 7v o o o 2 333 1 13 223 3 195 325 127 13s (Group 3) AmmmNo 14 15 1e 17 1s 19 2o o o o o o o 0 0 o o o o o o o o o o o 2 o 2 o o (Group. 1,)v

` o i o 0 3 o o o o o o n o o o o o o o 89 o o 2 5 o AnimalNo 14 15 16 11 18 10 20Y o 0 o o o o o o o 1 o o o o o o `o 0 o o o 2 21)V o Q Y 3 17 o CULTI'VATED AT THE TIME OF AUTPSY G VEEKSAFTER' of 6 animals died during l 4 the lnst vleeks ol experiment. :.Qindcates contamination. f

sulfate of Antiobiotic 23572 and dihydrostreptomycin sulfate were calculated by Litchfield and Wilcoxons method. As shown below, no significant diierence was found between them:

Sulfate o! Anti- Dihydrostrepto- `biotic 23572, mgJkg. rnycin sulfate,

mia/kg.

Intravenous injection. 230 219- 285) 210 215- 238) subcutaneous injectie 2,000 -(l,7l02.310) 1,5100 (1,450-2,250) )intraperitoneal injection.. 1,900 (l,50l)2,390) 1,700 (1,430-2,020)

(C) Antz'bacterial spectrum The sulfate of Antibiotic 23572 was compared with the commercial dihydrostreptomycin sulfate in weight for causing complete inhibition of test micro-organisms t0 give the following results:

ANTIBACTERIAL SPECTRUM The foregoing procedures are described in more detail in the following examples, but these are given by way of illustration andl not for the purpose of limitation. In these examples, the antibiotic potency is assayedby the cylinder-plate method using Bacillus subtilis (PCI 219) as thetest organism, except when theA method'for assay. is. particularly noted:

Example 11 Percent by weight Glucose 2.0 Meat extract 0.6 Peptone 1.0 Table salt 0.6 Calcium carbonate 0.6

500 liters of a culture medium=containing,thepabove Percent Gnpnqn components was sterilized by heating in a cultivating ves- Men umm., 0 6 sel. A strain belonging to Streptomyces liimii'diis was Peotnne.- 1.0 inoculated and cultivation was conducted at 27-28 C. 3:2 for 96 hours under aerobic conditions with agitation, 5 (4) Starch-m 0.5 whereupon the fermentation broth exhibited the following geelfr 32g antibacterial potency: 500 mcg/ml. against B. subtilis. gettone-.- (11g non n Example NaCl 0.3

Percent by weight com Steep liquor 3.0 l All percentages in this and in the following percentages Starch 3 0 are by weight. i Peptone 0.5 Example 4 Potassium dihydrogen phosphate 0.1 Basaimedium; Magnesium sulfate 0.05 starchl pereni 3 0 `Calcium carbonate 0.3 l K2HP04 d0 0,1 50 cc. of a culture medium containing the above com- MgSO47H2O -d0 0-05 ponents was sterilized in a shaking ask by a conventional VCaCOS .-f do-",- 0'3 method. A strain belonging to Szreptomyces lzumidus PH 7-0 was inoculated and cultivated at 27- l C. for 4 days ,20 with shaking, whereupon antibacterial potency of the om Steep liquor and Soybean meal Were added at fermentation broth reached 1200 mcg/m1, against various rates as nitrogen sources to the basal medium,` subtilis.' 'i v and strain H-lZO of Streptomyces liumidiis was inoculated The Strain employed in Examples 1, 2 and 9 is Sfrain and cultivated to obtain the results shown in the table 23572 (original) belonging to Strepiomyces hum'dus, 425 below: n respectively.

Example 3 Nitrogen source Maximum Time for Strain H-lwhich-belongs to Streptomyces humia'us, poteicy of obtaining wascultivated on various screening culture media. The Comme Soybean mpl? results are shown inthe following table. Y Each culture, lfijlont ggi nrlnyf hours shown in Examples 3-8, was conducted with shaking at peice p cg' o l 26-28 C. using 50 cc. of the medium in 300 cc. flask. 3 0 1.0 468 120 3.0 2(88 144 Maximum v'Ilme loi'V 35 M di rpgimiy """ahmg sfo o5 312 144 o um o vtroina 'rum strertopotency, 3'0 l 1' o 968 144 t mycin, hours mcg/ml l Soybean meal was hydrolyzed by heating at 100 C. for 1 hour with 10 times its volume of 5% hydrochloric acid. .glare hufig ggg gg 40 UCOFE OLll Ol'l starch honiiinn plus` Cacot, 3.0% 211 144 Example 5 Glucose bouillon plus CaCOa, 3.0% 286 120 Basal medium; (1).. 227 12o (m 246 Starch percent-- 3.0 (3) 329 120 K HPO do O 1 4 242 12o 45 2 4 l 315 Y 120 MgsoiJHzo do 0.05 Caco... .dn- 0.3 Percent PH 7.0 (1) Glucose 1.0 l

'ggllggnm (lig 50 Strain H-120 of Streptomyces humi'dus was cultivated Iagio. g-g on various media which contained, beside the basal com- (2) cmsipuqun, 320 i, ponents,corn steep liquor and soybean meal as pringgtcor-- 3:2 jcipalmnitrogen sources, and other organic or inorganic KiiiqPof' 0b; .nitrogen sources. In some cases, soybean meal was omitlfgfno 6 3 l55 ted; Thel'results are shown in the following table:

Nitrogen source Maximum Time for potency of obtaining Soybean dihydromaximum Corn steep meal streptopotency, liquor. (1i f`i-hy- Others, percent iiiyoin, hours percent drolyzate). mcg/ml. percent 3.0 1. o Myceiiiim of 23572 (wet). 2. o 66o 144 3.o 1.o 'Hivos 0.1 326 12o Sailer si it it 31 1- 0 iLard 011.2..12 i.' o 3.0 1.o 0.1 411 12o 3.o 1.o 0.1 60o 9s 3.o 1.o 0.1 327 120 3.o 1.ok 0.1 39e 144 3.o 1.o 50o 12o 3.o 1.o 1,000 144 1.0 2.0 v. 3o 168 l Caseiu was hydrolyzed at C. for 1 hour with about 10 times it's volume o1 5% HCL.

noem-oe dextrin, but the composition of inorganic salts was changed variously. The results are shown in the table below.

Basal medium:

Dextrin percent 3.0 Corn steep liquor -do 3.0

' Example 6 'in various .ways were'cultiv'ated on the above medium. Basal medium: The Aresults are shown in the table below:

Corn steep liquor percent-- 3.0 f Soybean meal (HCl-hydrolyzate) do 1.0 Mutats Maximum Timm K2HPO4 --d0 o-l 5 piitny of obtniining y lO- 1113K mum MgSO4`7H2O "do-' 0'05 Induced Mcthodsfor streptopotency, CaCOa --d0-- 03 Strains trommatation l mycin. hours PH 7.o mcg/mi. l

Strain Q-97 of Streptomyces humidus was cultivated 23572mgmal) 660 144 on the basal medium 1n which the carbon source was B-o l?) changed variously. The results were as shown in the 792 120 following table: 433 144 1.220 144 l' lit Maximum Time tor f potency of 1 obtaining 15 1, Carbon source dihydromaximum V 931 144 streptopotency, 574 120 mycin,4 Y hours 975 120 mcg/ml. I 1,235 1.44 s@ a: Percent 2 3.o 1,127 12o 0 l 374 120 1. o soo 12o '085 120 3.o 844 12o 1.0 592 `12o 3.0 440 120 l Methods for mutatlon: means mono-spore separation, D moans 3 0 1 050 .144 inoculation of ythe stmin on a dihydrostrcptomycin-contalning medium, 3:0 90 l 9B UV means ultraviolet-ray radiation ofthe strain.

1333 i'14 i U E 1 9 xamp e 3.o l1,870 144 3. o 1, 39o 144 Medlllm Suero 3-0 63 144 Glucose ---v..- percent..- 2.0 Meat extract do 0.6 Example 7 30 Peptone do.. 1.0

(l Strain Q-97 of Streptomyces humzdus was cultivated gag') d2 on various media, in which nitrogen sources were corn I?! 3 7'0 steep liquor and soybean meal, and carbon source was p AY strain belonging to Streptomyces humidus was subjected to aerobic tank culture for 96 hours. To 700 l. ofthe culture filtrate (pH: 8.5, potency: u./cc. (dilution unit) against E. coli) was added 7 kg. (1%) of active carbon, and the mixture was stirred for 30 min- Soybean meal (HCl-hydrolyzate) do 1.0 40 utes to adsorb the active compound. The active carbon was eluted with 10 times its volume of methanolic hydro- Inorgan'c salts Maximum 'Fimo for potency ol obtaining dihydro marlmum` K2HPO4, MgSOMFMO, CaCO, Others streptomycin, potency, hours percent percent percent mcgJml.

........ 0. 05 0. 3 717 120 0. 1 0.3 675 120 0` 1 0. 05 0. 0 1, 230 Y 144 0. 1 0.05 0. 3 (l) T00 144 0.1 0.05 1. 0 1,260V 120 0. 1 0.05 0.3 ZnSOrJHO, 0.001%. 639 144 0. 1 0.05 0.3 MnSO4.7H:O, 0.002% 555 144 0. 1 0.05 0.3 FeSO4JHzO, 0.002% 1, 105 120 0. l 0.05 0.3 NagSO (anhydrous), 0.1%.. 1.099 144 0. 1 0. 05 0. 3 CuSO4.5H;O, 0.002% 1, 038 120 dro 1 Neutralization oi h xld ydrolyznte ol' soybean' meal and adjustment ot pH were conducted with potassium hygenchlorde for 30 minutes at pH 2.0 to obtain 140 1. of the eluate (potency: 100 u./cc. (dilution unit)). The elution was repeated again to obtain ca. l. of the eluate (potency: 35 u./cc. (dilution unit)). The combined eluates were neutralized with N-NaOH to pH 6.5 and`concentrated in vacuo at low temperature to about 300 cc. During the process the separated sodium chloride is removed now and then. 3 l. of anhydrous acetone is added to the concentrated solution, and the precipitated active compound was dried in vacuo to give white powder. The yield was 147 g. or 60% (potency: 100

Mutants obtainedl by treating Streptomyces humdus 17 u./mg. (dilution unit)). The product is negative to maltol reaction but positive to Sakaguchi reaction.

Mutants described in this specification except the mutants used in the preceding Example 8 are induced by such methods as follows: K

(i) Mono-spore separation of strain 23572 Y used in the preceding Example 8 gives strain 23572 W.

(ii) Inoculation of the strain Q-97 used in the preceding Example 8 on a dihydrostreptomycin-containing medium gives strain H-120.

The specimen yof strain 23572 has been filed with Institute for Fermentation, Osaka (IFO), a Japanese culture collection, and its deposit number is IPO-3520.

A specimen of the said strain 23572 has also been deposited with thev American Type Culture Collection, ATCC No. 12760.

What is claimed is:

1. A process for producing dihydrostreptomycin, which comprises cultivating a member selected from the group i consisting of a dihydrostreptomycin-producing strain of Streptomyces humidus and its natural mutants, induced mutants and variants in an aqueous nutrient-containing medium under aerobic conditions until substantial antibacterial activity due to accumulation of formed dihydrostreptomycin is imparted to said medium.

2. A process for producing dihydrostreptomycin, which comprises cultivating a member selected from the group consisting of a dihydrostreptomycin-producing strain of Streptomyces humidus and its natural mutants, induced mutants and variants in an aqueous nutrient-containing medium under aerobic conditions until the antibiotic is accumulated at a concentration of at least lOy/cc. in the resultant fermentation broth. Y

3. A process for producing dihydrostreptomycin, which comprises cultivating a member selected from the group consisting of a dihydrostreptomycin-producing strain of Srrepromyces humidus and its natural mutants, induced mutants and variants in an aqueous nutrient-containing medium under submerged aerobic conditions until suby18 stantial antibacterial activity due to accumulation of formed dihydrostreptomycin is imparted to said medium.

4. A process for producing dihydrostreptomycin, which comprises cultivating a member selected from the group consisting of a dihydrostreptomycin-producing strain of Streptomyces humdus and its natural mutants, induced mutants and variants in an aqueous nutrient-containing medium under submerged aerobic conditions until the antibiotic is accumulated at a concentration of at least 10'y/cc. in the resultant fermentation broth.

5. A process for producing dihydrostreptomycin, which comprises cultivating a dihydrostreptomycin-producing strain of Streptomyces humdus in an aqueous nutrientcontaining medium under aerobic conditions until substantial antibacterial activity due to accumulation of formed dihydrostreptomycin is imparted to said medium.

6. A process for producing dihydrostreptornycin, which comprises cultivating a dihydrostreptomycin-producing strain of Streptomyces humdus in an aqueous nutrientcontaining medium under aerobic conditions until the antibiotic is accumulated at a concentration of at least 107/cc. in the resultant fermentation broth.

7. A process for producing dihydrostreptomycin, which comprises cultivating a dihydrostreptomycin-producing strain of Streplomyces humdus in an aqueous nutrientcontaining medium under submerged aerobic conditions ata temperature of about 24 to 30 C. for about 3 to 8 days.

References Cited in the file of this patent UNITED STATES PATENTS 2,498,574 Peck Feb. 21, 1950 OTHER REFERENCES Langlykke et al. Ian. 3, 1950 i UNITED STATES PATENT OFFICE CERTIFICATE 0F CORRECTION Patent No. 2,931,756 AprII 5, 1960 Koiti Nakazawa et alo lt is hereby certified that error appears in the above I;

numbered patent requiring correction and that the said Letters Patent should rea-d as corrected belowo In the heading to the printed specification, between line 8 and 9, insert Application March 2l, 1957, .Serial No. 647,681 column l5, first table, column l, line. 4 thereof,

for "Glucouse" read Glucose --o Signed and sealed this 6th day of September 1960 (SEAL) Attest:

ERNEST W, SWIDER Attestng Officer ROBERT C. wATsoNf Comnssoner of Patents; 

1. A PROCESS FOR PRODUCING DIHYDROSTREPTOMYCIN, WHICH COMPRISES CULTIVATING A MEMBER SELECTED FROM THE GROUP CONSISTING OF A DIHYDROSTREPTOMYCIN-PRODUCING STRAIN OF STREPTOMYCES HUMIDUS AND ITS NATURAL MUTANTS, INDUCED MUTANTS AND VARIANTS IN AN AQUEOUS NUTRIENT-CONTAINING MEDIUM UNDER AEROBIC CONDTIONS UNTIL SUBSTANTIAL ANTIBACTERIA ACTIVITY DUE TO ACCUMULATION OF FORMED SIHYSROSTREPTOMYCIN IS IMPARTED TO SAID MEDIUM. 